PROJECT SUMMARY The serotonin (5-HT) neurotransmitter system comprises at least 14 distinct receptors that share topologically similar orthosteric sites, a fact that contributes to the historically difficult goal of sub-type selective modulation. Allosteric modulation of G protein-coupled receptors (GPCRs) can yield drug candidates for previously elusive receptors through targeting a structurally divergent allosteric site. Evidence suggests that dysregulated signaling of the 5-HT2C receptor underlies pathological processes implicated in numerous mental health disorders, including cocaine use disorder, which is without an FDA-approved medication. Interestingly, reduced 5-HT2C receptor signaling plays a modulatory role in stimulating relapse-related phenotypes in preclinical models of cocaine addiction, and conversely, rescuing this dysfunction may provide clinical utility to promote and maintain abstinence. We hypothesize that the rational design of novel 5-HT2C receptor positive allosteric modulators (PAMs) and optimization of 5-HT2C receptor PAM lead compounds will serve as a useful strategy to rescue pathologically decreased 5-HT2C receptor signaling and, ultimately, suppress relapse-related behavioral phenotypes. To this end, 4-alkylpiperidine-2-carboxamides were designed and synthesized in a PAM hit-to-lead discovery effort. The work resulted in the discovery of two novel lead compounds, which displayed subtype- selective functional enhancement of the 5-HT2C receptor, and lacked intrinsic agonism, in vitro. Leads, CTW0415 and CYD-6-2-1, exhibited favorable in vitro and in vivo pharmacokinetics, while a broad-panel GPCR screen confirmed selectivity. In addition, these leads maintain adequate lead-like physicochemical characteristics and represent ideal molecules upon which to build our lead optimization efforts. Our iterative strategy for 5-HT2C receptor PAM lead optimization will enable robust structure activity relationship (SAR) and will be accomplished by 1) rational design and synthesis of novel 5-HT2C receptor selective PAMs; and 2) biological characterization in a cell-based 5-HT2C receptor functional assay and pharmacological characterization in a radioligand binding assay. The proposed work will advance our knowledge of 5-HT2C receptor positive allosteric modulation and provide insight towards PAM mechanism. Additionally, the current PAM lead compounds require activity enhancements via optimization to enable development of preclinical candidates. The completion of this proposal will foster technical expertise and scientific intuition, ultimately enabling the applicant to achieve the goal of becoming an academic chemical biologist studying drug use disorders and the neuropharmacology of drugs of abuse. The applicant will gain experience in medicinal, organic and computational chemistry; neuroscience; molecular and cellular pharmacology; GPCR targeting experimental design; and the fundamentals of drug discovery in allosteric modulation, neurobiology and the psychostimulant addiction fields. Taken together, the present studies represent a high impact approach towards the discovery of neurotherapeutics for cocaine use disorder, and will generate novel tools to elucidate 5-HT2C receptor function and potential therapeutic candidates.